Technical Field
The present invention is generally related to oligonucleotide compounds (oligomers) useful as antisense compounds, and more particularly to oligomer compounds comprising modified intersubunit linkages and/or terminal groups, and the use of such oligomer compounds in antisense applications.
Description of the Related Art
Antisense oligomers are generally designed to bind in a sequence specific manner to DNA or RNA regions and alter the expression of disease-causing proteins. Requirements for successful implementation of antisense therapeutics include (a) stability in vivo, (b) sufficient membrane permeability and cellular uptake, and (c) a good balance of binding affinity and sequence specificity. Many oligonucleotide analogues have been developed in which the phosphodiester linkages of native DNA are replaced by other linkages that are resistant to nuclease degradation (see, e.g., Barawkar, D. A. et al., Proc. Na't'l Acad. Sci. USA 95(19):11047-52 (1998); Linkletter, B. A. et al., Nucleic Acids Res. 29(11):2370-6 (2001); Micklefield, J., Curr, Med, Chem, 8(10):1157-79 (2001)). Antisense oligonucleotides having other various backbone modifications have also been prepared (Crooke, S. T., Antisense Drug Technology: Principles, Strategies, and Applications, New York, Marcel Dekker (2001); Micklefield, J., Curr, Med, Chem, 8(10):1157-79 (2001); Crooke, S. T., Antisense Drug Technology, Boca Raton, CRC Press (2008)). In addition, oligonucleotides have been modified by peptide conjugation in order to enhance cellular uptake (Moulton, H. M. et al., Bioconjug Chem 15(2):290-9 (2004); Nelson, M. H. et al., Bioconjug. Chem. 16(4):959-66 (2005); Moulton, H. M. et al., Biochim Biophys Acta (2010)).
The performance of such nucleic acid analogues as antisense or antigene drugs has been hampered by certain characteristics of the various analogues. For example, analogues with negatively charged linkages, including phosphorothioate-linked analogues, suffer from considerable electrostatic repulsion between the negative charges of the oligomer and the DNA or RNA target. The phosphorothioates also exhibit non-specific binding to other cellular components such as proteins. These attributes limit the therapeutic effectiveness of antisense oligomers comprised of native RNA, native DNA, and negatively charged analogues (Crooke, S. T., Antisense Drug Technology: Principles, Strategies, and Applications, New York, Marcel Dekker (2001); Crooke, S. T., Antisense Drug Technology, Boca Raton, CRC Press (2008)). The nonionic methylphosphonate-linked oligonucleotide analogues can be transported into cells by passive diffusion and/or fluid phase endocytosis, but their use is hampered by stereoisomeric complexity and poor solubility (Crooke, S. T., Antisense Drug Technology: Principles, Strategies, and Applications, New York, Marcel Dekker (2001); Micklefield, J., Curr, Med, Chem, 8(10):1157-79 (2001)).
Several groups have reported the synthesis of positively charged oligonucleotides (Bailey, C. P. et al. Nucleic Acids Res. 26(21):4860-7 (1998); Micklefield, J., Curr, Med, Chem, 8(10):1157-79 (2001); Egli, M. et al., Biochemistry 44(25):9045-57 (2005)). For example, a class of guanidinium linked nucleosides (designated DNG), formed by replacement of the phosphate linkages in DNA and RNA by achiral guanidino groups, has been reported (Dempcy, R. O. et al., Proc. Nat'l Acad. Sci. USA 91(17):7864-8 (1994); Dempcy, R. O. et al., Proc. Nat'l Acad. Sci. USA 93(9):4326-30 (1996); Barawkar, D. A. et al., Proc. Na't'l Acad. Sci. USA 95(19):11047-52 (1998); Linkletter, B. A. et al., Nucleic Acids Res. 29(11):2370-6 (2001)). Oligomers linked with positively charged methylated thiourea linkages have also been reported (Arya, D. P. et al., Proc. Nat'l Acad. Sci USA 96(8): 4384-9 (1999)). Replacement of some of these linkages with neutral urea linkages has been reported to reduce the tendency of such positively charged oligomers towards non-sequence-specific binding (Linkletter, B. A. et al., Bioorg. Med. Chem. 8(8):1893-901 (2000)). Morpholino oligomers containing (1-piperazino) phosphinylideneoxy and (1-(4-(ω-guanidino-alkanoyl))-piperazino) phosphinylideneoxy linkages have been described previously (see e.g., WO2008036127).
Although significant progress has been made, there remains a need in the art for oligonucleotide analogues with improved antisense or antigene performance. Such improved antisense or antigene performance includes; stronger affinity for DNA and RNA without compromising sequence selectivity; improved pharmacokinetics and tissue distribution; improved cellular delivery and reliable and controllable in vivo distribution.